Ion channels and transporters control the movement of charged ions across cell membranes. In the heart, the coordinate activities of these proteins regulate the transmembrane electrochemical gradient to control depolarization/repolarization, and thus cardiac excitability. Normal function of ion channels/transporters requires defined biophysical properties as well as precise expression, organization, and regulation in defined membrane domains. Our recent findings support a new basis for arrhythmia based not on mutations which affect channel biophysics, but instead on mutations to proteins which are required for proper expression and local regulation of ion channels and transporters at excitable membranes. Ca2+/calmodulin-dependent protein kinase II (CaMKII) is a multifunctional signaling molecule with critical roles in cardiac physiology and heart disease. Similar to protein kinase A (PKA) and protein kinase C (PKC), CaMKII regulates the phosphorylation status of a host of target substrates throughout the cell. However, in striking contrast to PKA- and PKC-dependent local signaling, little is known regarding local CaMKII targeting in myocytes. In fact, while a handful of CaMKII-binding partners have been proposed to regulate local signaling, no CaMKII-targeting proteins have been validated in animal models. Our preliminary data support a novel molecular mechanism for CaMKII targeting to the myocyte intercalated disc. bIV spectrin, an actin- and ankyrin-G-associated molecule, associates with CaMKIId both in vitro and in vivo. bIV spectrin, previously only studied in the nervous system, is expressed in heart, associates with ankyrin-G, and targets CaMKII to the myocyte intercalated disc to directly regulate local voltage-gated Na+ channel activity. Notably, we have identified a previously elusive CaMKII phosphorylation site on Nav1.5 (S571) that regulates channel activity. A targeted mouse model deficient in the bIV spectrin CaMKII-binding motif lacks intercalated disc CaMKII expression resulting in reduced Nav1.5 S571 phosphorylation, altered Nav channel activity, action potential abnormalities, and whole animal cardiovascular phenotypes. These preliminary data strongly support bIV spectrin as a previously unrecognized in vivo CaMKII-targeting protein, as well as our central hypothesis that the bIV spectrin-based intercalated disc complex provides critical signaling and structural roles for normal cardiac function. Furthermore, these data strongly suggest that targeted inhibition of the bIV spectrin/CaMKII interaction would serve as a novel, and highly specific mechanism to suppress persistent INa in the heart to treat arrhythmia. While this proposal will primarily focus on the role of bIV spectrin-targeted CaMKII in regulating Nav1.5, we hypothesize that the bIV spectrin complex will play a broader role in local signaling and structural regulation at the myocyte intercalated disc. We predict that these findings will provide the first data for local CaMKII targeting in vivo, provide the first data for the role of CaMKII and Nav1.5 S571 for cardiac INa regulation in health and disease, define the role of ankyrin-G and bIV spectrin in cardiac signaling, membrane structure, and function, and finally define a novel and potentially therapeutic mechanism to suppress persistent INa in heart to treat arrhythmia. PUBLIC HEALTH RELEVANCE: Normal function of ion channels/transporters requires defined biophysical properties as well as precise expression, organization, and regulation in defined membrane domains. CaMKII is a multifunctional signaling molecule with critical roles in cardiac physiology and heart disease. Our studies will provide the first data for local CaMKII targeting in vivo, provide the first data for the role of CaMKII and Nav1.5 S571 for cardiac INa regulation in health and disease, define the role of ankyrin-G and bIV spectrin in cardiac signaling, membrane structure, and function, and finally define a novel and potentially therapeutic mechanism to suppress persistent INa in heart to treat arrhythmia.